A fundamental question in developmental neurobiology is how a limited number of growth factors coordinate the establishment of precise neuronal circuits during nervous system development. Neurotrophins provide one of the best examples of target-derived developmental cues regulating neuronal survival, axonal and dendritic growth and synaptogenesis. An intriguing question in neurotrophin research is how a very large number of biological events are triggered by such a limited set of neurotrophins and their receptors. We previously reported that two different neurotrophins, Nerve Growth Factor (NGF) and Neurotrophin-3 (NT-3), employ the same TrkA receptor to promote sequential stages of axonal growth during sympathetic nervous system development. NT-3, secreted from the vasculature along the trajectory of projecting sympathetic axons, promotes early axon outgrowth. However, NGF derived from peripheral targets is required for final innervation of end- organs. How does a common TrkA receptor respond to two different neurotrophins to facilitate distinct phases of axon growth? We identified a calcineurin-dependent TrkA endocytic pathway that is critical for NGF-, but not NT-3-dependent trophic functions. Calcineurin is a calcium-responsive phosphatase that influences diverse aspects of neuronal development by translating small changes in intracellular calcium levels to morphological and transcriptional changes. Thus, the overall goal of this proposal is to test the hypothesis that the two neurotrophins, NGF and NT-3, differentially regulate TrkA signaling and trafficking to promote distinct stages of sympathetic axon growth. To this end, we will employ biochemical assays to identify TrkA signaling pathways that allow target-derived NGF and NT-3 to differentially activate calcineurin in sympathetic neurons. Using mutant mice lacking calcineurin or its downstream target, the endocytic GTPase, dynamin1, we will test the hypothesis that endocytosis of TrkA receptors is specifically required for NGF, but not NT-3-mediated sympathetic axonal growth in vivo. We will also define the mechanisms by which TrkA endocytosis promotes axonal growth. Together, these studies will provide novel insights into how two target-derived neurotrophins signal via a common TrkA receptor to cooperatively regulate axonal growth during neuronal development, as well as provide the foundation for addressing the role of these axonal growth programs in mediating regenerative growth of adult neurons following injury or disease. PUBLIC HEALTH RELEVANCE: The initiation, extension and targeting of axons during development underlies the formation of precise neuronal circuits, necessary for physiological and cognitive functions. Axon degeneration is a hallmark of spinal cord injuries and almost all neurodegenerative disorders. Thus, it is essential to gain a better understanding of molecular mechanisms that promote axonal growth during nervous system development, with the goal to apply this information to axonal repair strategies during neurological disorders or injury. This proposal aims to identify a critical receptor trafficking mechanism by which neurotrophic growth factors promote axon growth during development. The proposed research has the potential to impact future neurotrophin-mediated therapeutic strategies to enhance regenerative growth following injury and disease.